Nos gustaría dar las gracias a L. Evans, B. Granger, Rissling D., Sim Z., A. Garcia, Hayhurst K. y D. Kuch de asistencia en el campo. K. Galbreath amablemente proporcionaron muestras de hígado pika del valle de Bella Coola. Gracias a A. Gonçalves da Silva y K. Larsen para discusiones interesantes que contribuyeron al diseño de este método de muestreo genético no invasivo. Este trabajo fue financiado por las Ciencias Naturales e Ingeniería de Investigación de Canadá Discovery, y UBC Okanagan becas de investigación individuales de marzo A Fondo Nacional Suizo Fundación de Becas de Doctorado PBSKP3_128523 apoyado PH. Este estudio se realizó siguiendo el protocolo de cuidado de los animales de la Universidad de British Columbia (número de certificado: A07-0126).

1. Pelo trampa Antes de la creación de una trampa, una ubicación ideal tiene que ser determinada en el hábitat del pika o talud. Esto incluye montones de heno, que son escondites de vegetación que los animales se acumulan en verano, así como heces dulce que se encuentra en los sitios de letrina. Tiras de cinta de embalaje (10-50cm de longitud) se acumulan para proporcionar una superficie de 360 ​​° pegajoso y se organizan de una manera similar a una red para envolver a la entrada de la pila de heno la pika (Fig. 1), o sitio de la letrina. Dependiendo de la configuración de las rocas, un trozo de hilo de pescar se puede utilizar para apoyar la estructura de la trampa de pelo, pero a menudo no es necesario cuando las entradas son muy pequeñas (&lt;30cm de diámetro), una descripción completa de la utilización de línea de pesca se puede encontrar en Henry y Russello (2010) 3. Trampas de pelo son verificados siempre que sea posible, las muestras de pelo y depositados en la cinta (Fig. 2) se recogen y se etiqueta. Una vez transportados al laboratorio, los pelos se eliminan de la cinta adhesiva con una pinza estéril y se transfirieron a tubos criogénicos y almacenadas a -20 ° C hasta su posterior manipulación. Las muestras de cabello se agrupan a lo largo de un lazo del pelo se considera que pertenecen a una sola persona, mientras que las muestras agrupadas de forma independiente se supone que pertenecen a diferentes personas. En este último caso, el cabello se coloca en tubos diferentes. Estos supuestos más tarde se puede probar por medio de huellas de ADN y los cálculos de la probabilidad de identidad basado en microsatélites datos genotípicos. 2. Extracción de ADN Puesto que el pelo pika es muy delgada y contiene una bombilla de raíz pequeña, hemos demostrado que un mínimo de 25 pelos están obligados a obtener cantidades suficientes de ADN para la buena calidad de amplificación de PCR abajo 3. Se utilizó el sistema IQ ADN (Promega, Madison, WI, EE.UU.) y una versión ligeramente modificada de las instrucciones del fabricante para el aislamiento de ADN de muestras de cabello. En primer lugar, la muestra de cabello se hace girar en una centrífuga de micro-con el fin de evitar la pérdida de cabello, mientras que la apertura del tubo. A continuación, la solución de incubación se prepara mezclando 80 l de solución de incubación con 10μl de la TDT (1M) y 10μl de proteinasa K (18ng/ml) para dar lugar a una concentración final de 0,1 M TDT y 1.8ng/ml de proteinasa K. incubación solución (100 l) se añade a la muestra y se incuba a 56 ° C durante 1 hora. Mientras tanto, la solución de lisis se prepara añadiendo 1 l de DTT (1M) por cada 100 l de tampón de lisis, lo que resulta en una concentración final de 0,01 M de TDT. Solución de lisis preparado (200 l) se añade a la muestra, junto con 7 l de ADN de resina IQ, agitaron durante 3 segundos a alta velocidad y se incuban a temperatura ambiente durante 5 minutos. La muestra se agitaron durante 2 segundos y se inserta en el soporte magnético, donde la separación de partículas magnéticas de la solución se presenta. La solución restante se aspira y se desecha, teniendo cuidado de no molestar a los pellets de resina magnético. La muestra se trata con 100 l de la solución de lisis preparado, vortex y se devuelve al soporte magnético cuando la separación se produce de nuevo. El tampón de lisis que se aspira y se desecha, y este paso se repite tres veces con un tampón de lavado (100 l). La muestra posteriormente se deja secar en el soporte magnético durante 15 minutos. Una vez seco, 100 l de tampón de elución se añade a la muestra y se incuba a 65 ° C durante 5 minutos. La muestra se agita y se inserta en el soporte magnético. La solución que ahora contiene el ADN eluido se transfiere a un tubo Eppendorf de 1,5 ml y almacenadas a -20 ° C hasta más manipulaciones. ADN de muestras de hígado fue extraído utilizando también el sistema de inteligencia del ADN y utilizado como control positivo de amplificación de PCR. 3. Amplificación por PCR El ADN obtenido de nuestro lazo de pelo no invasiva y las muestras de hígado fueron utilizados para amplificar un conjunto de uso común de marcadores moleculares (microsatélites, AFLP, mitocondrial citocromo B y ZFX / ZFY marcador de determinación del sexo). PCR se realizó con un Veriti termociclador (Applied Biosystems, Foster City, CA, EE.UU.) en un volumen de 25 l que contiene: 10 - 20 ng de ADN, 0,5 mM de cada primer, 10 mM Tris-HCl (pH 8,3), 50 mM KCl, 1,5 mM de MgCl 2, dNTPs 200 mM, a 10 μ de albúmina sérica bovina (BSA; New England Biolabs, Ipswich, MA, EE.UU.) y 0,5 U AmpliTaq DNA polimerasa de oro (Applied Biosystems). Parámetros de los ciclos de los loci microsatélites se han optimizado mediante un programa de ciclos de touchdown (10 min a 95 ° C, 35 ciclos a 95 ° C durante 30 s, 30 s recocido, y 45 s a 72 ° C, seguida de una etapa final a 72 ° C durante 10 min). La temperatura de hibridación redujo en un 1 ° C por cada ciclo de 60 a 55 ° C hasta llegar al sexto ciclo, momento en el que los 29 ciclos restantes continuaron a 55 ° C. Parámetros de los ciclos de los fragmentos mitocondrial fueron descritos como más arriba, pero consistió en 35 ciclos con un recocidotemperatura de 50 ° C. Amplificación de fragmentos polimórficos se llevó a cabo siguiendo el protocolo de los genomas de vertebrados descritos por Bonin 4. Por último, sexado molecular se ha realizado mediante la técnica PCR-RFLP de ZFX / ZFY loci con la digestión de la enzima de restricción HinfI 5. Los productos de PCR de la microsatélites, AFLP y las secuencias del citocromo B se realizaron en un analizador genético ABI 3130xl (Applied Biosystems), mientras digiere ZFX / ZFX fragmentos se llevaron a cabo junto a una escalera de 100 pb (New England Biolabs) en un 3% en gel de agarosa que contiene 2,5% en gel de ADN SYBR seguro. mancha (Invitrogen, Carlsbad, CA, EE.UU.) y se visualizan mediante un sistema RED de imagen personal gel (Alpha Innotech, San Leandro, CA, EE.UU.) Microsatélites y AFLPs se visualizaron utilizando GeneMapper v3.7 (Applied Biosystems) y el cromatograma de ADN mitocondrial se visualizan mediante Sequencher v4.7 (Código Gene Corporation, de Ann Harbor, MI, EE.UU.). 4. Los resultados representativos El ADN extraído de muestras de cabello obtenido usando nuestro lazo no invasiva se utiliza para amplificar por PCR de varios tipos de marcadores moleculares. Como base de comparación, utilizando ADN extraído de muestras de hígado se amplificó junto con muestras de nuestro cabello. Nuclear loci microsatélites fueron amplificados con éxito, tanto para el cabello y las muestras de hígado (Fig. 3). A pesar de la intensidad de la señal fue mayor cuando se utilizan las muestras de hígado, esto no tuvo un efecto negativo en la puntuación genotipo (Fig. 3). Resultados similares se obtuvieron cuando se utiliza AFLPs (Fig. 4), secuencias de ADN mitocondrial (Fig. 5), y el marcador ZFX / ZFY sexado molecular (Fig. 6). <img src="/files/ftp_upload/2791/2791fig1.jpg" alt="Figura 1"/> Figura 1. Un ejemplo de una trampa de pelo no invasiva creado en una pila de heno. Tiras enrolladas de cinta adhesiva transparente se organizan de una manera similar a una red para cerrar la entrada a la pila de heno. Un pedazo de hilo de pesca se utiliza aquí para ofrecer ayuda adicional a la trampa de pelo. <img src="/files/ftp_upload/2791/2791fig2.jpg" alt="Figura 2"/> Figura 2. Una trampa de pelo éxito que contiene una gran cantidad de pelo pegado a la cinta de embalaje. <img src="/files/ftp_upload/2791/2791fig3.jpg" alt="Figura 3"/> Figura 3. Un cromatograma representativo de microsatélites nucleares (Ocp6) 8 como se muestra en GeneMapper v3.7 (Applied Biosystems). El cromatograma superior se obtuvo mediante la amplificación de ADN de tejido del hígado, y es heterocigoto (354/358) en este lugar. El cromatograma inferior representa una persona diferente exhibiendo un genotipo heterocigoto (358/362), basado en ADN extraído de pelo. Mientras que la señal del cromatograma de la muestra de cabello tiene una intensidad menor que la muestra de hígado, anotando el genotipo no se ve afectada por esta diferencia en la señal. <img src="/files/ftp_upload/2791/2791fig4.jpg" alt="Figura 4"/> Figura 4. AFLP un cromatograma representativo (E31T32) como se muestra en GeneMapper v3.7 (Applied Biosystems). El cromatograma superior se obtuvo mediante la amplificación de ADN de tejido del hígado, el cromatograma inferior representa el ADN extraído de pelo. <img src="/files/ftp_upload/2791/2791fig5.jpg" alt="Figura 5"/> Figura 5. Un representante cromatograma secuencia de ADN mitocondrial (citocromo b) como se muestra en Sequencher v4.7 (Gene Código de Sociedades). El cromatograma superior se obtuvo mediante la amplificación de ADN de tejido del hígado, el cromatograma inferior representa el ADN extraído de pelo. <img src="/files/ftp_upload/2791/2791fig6.jpg" alt="Figura 6"/> Figura 6. Un marcador representante sexado molecular (ZFX / ZFY) amplificada por el hígado y las muestras de cabello. Este enfoque se basa en la observación de que el cromosoma Y tiene un sitio de restricción HinfI en esta región del cromosoma X no tiene. Por lo tanto, las hembras producen dos migración co-fragmentos de 432 pb, mientras que los machos producen fragmentos de 432 pb, 261 pb y 171.

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Muestreo genético no invasivo se ha convertido en una alternativa atractiva a los tradicionales métodos de captura en vivo por varias razones. En primer lugar, discretamente recolección de material biológico (por ejemplo, heces, pelos, plumas, saliva y moco) de las poblaciones silvestres, los investigadores pueden estudiar estas especies sin alterar, manipular, o incluso la observación, por lo tanto reducir los riesgos para los animales y los investigadores. En segundo lugar, el muestreo genético no invasivo permite a los biólogos para estudiar las poblaciones de especies evasivas y raras, una tarea que puede resultar difícil con más tradicional en vivo que atrapa enfoques 6. Y en tercer lugar, NGS potencialmente puede aumentar tamaño de las muestras mediante la reducción de molestias a los animales, los esfuerzos de muestreo, y los costos, lo que ayuda a minimizar los sesgos en las estimaciones de los parámetros de la población 7. Este último punto puede ser crucial cuando se trata de especies amenazadas, ya que las estimaciones sesgadas de los parámetros de la población puede dar lugar a una gestión inadecuada. En el artículo de vídeo actual se describe un método simple y novedoso, barato y no invasivo para la muestra esquivo pequeños mamíferos, con el estadounidense Pika como un ejemplo. Se demuestra que el ADN extraído de pelo funciona de forma similar al ADN extraído de muestras de hígado con respecto a los microsatélites, AFLP, mitocondrial y marcadores de determinación del sexo, por lo que este método de muestreo no invasivo en una alternativa atractiva para vivir de muestreo trampas o letal. En general, prevemos que este método sea útil en la etapa de recolección de datos de los estudios de genética de la población y del comportamiento de las especies de pequeños mamíferos raros o difícil de alcanzar, mientras que se minimiza el impacto sobre los organismos en estudio.

Pelo trampa: <ul><li> Rollos de cinta adhesiva transparente (3M, St. Paul, MN, EE.UU.) </li><li> La pesca con caña (10 libras) </li><li> Criogénica del tubo (2 ml) </li><li> Pinzas estériles </li><li> Dewar de nitrógeno líquido (opcional) </li></ul> Extracción de ADN: <ul><li> ADN IQ System (Cat. # DC6701; Promega) con el tejido y el kit de extracción de pelo (Cat. # DC6740, Promega). </li><li> MagneSphere soporte tecnología de separación magnética (Cat. # Z5342, Promega) </li><li> Microcentrífuga </li><li> Horno de hibridación o el agua del baño </li><li> Los tubos Eppendorf de 1,5 ml </li></ul>

a noninvasive hair sampling technique to obtain high quality dna from elusive small mammals

No invasiva de los métodos de muestreo genético son cada vez más importante para el estudio de poblaciones de vida silvestre. Un número de estudios han reportado el uso técnicas no invasivas de muestreo para investigar la genética de poblaciones y la demografía de las poblaciones silvestres 1. Este enfoque ha demostrado ser especialmente útil cuando se trata de especies raras o difícil de alcanzar 2. Aunque algunos de estos métodos han sido desarrollados para el pelo de la muestra, las heces y otros materiales biológicos de los carnívoros y los mamíferos de tamaño mediano, que han permanecido sin probar en difícil de alcanzar pequeños mamíferos. En este vídeo, se presenta una novela, la trampa de pelo barato y no invasivo destinado a un elusivo animal pequeño, el pika americano (Ochotona princeps). Se describe la configuración general de la trampa de pelo, que consiste en tiras de cinta de embalaje dispuestos de una manera similar a una red y se colocan a lo largo de las rutas de viaje en el hábitat de los pikas. Se ilustra la eficacia de la trampa en la recolección de una gran cantidad de pelo que puede ser recogido y llevado al laboratorio. A continuación, mostrar el uso del sistema de ADN IQ (Promega) para aislar el ADN y demostrar la utilidad de este método para amplificar comúnmente utilizados como marcadores moleculares microsatélites nucleares, polimorfismos de longitud de fragmentos amplificados (AFLP), secuencias mitocondriales (800bp), así como una marcador de sexado molecular. En general, se demuestra la utilidad de esta trampa de pelo nuevo no invasivo como una técnica de muestreo para los biólogos de poblaciones de vida silvestre. Creemos que este enfoque se aplica a una variedad de pequeños mamíferos, la apertura de áreas de investigación dentro de las poblaciones naturales, mientras que minimiza el impacto en los organismos de estudio.

Watch this Scientific Journal Video about A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals at JoVE.com

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Se presenta un método de muestreo no invasivo para recoger muestras de cabello de manera eficiente difícil pequeños mamíferos, como se muestra en la pika estadounidense. Demostrar la utilidad de este método de extracción de ADN de muestras de cabello y amplificar varios tipos de marcadores moleculares utilizados en los estudios de la ecología de la fauna silvestre y la conservación.

Un cabello no invasiva técnica de muestreo para obtener ADN de alta calidad de esquiva Pequeños Mamíferos

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

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Biology

20.7K vistas.  University of British Columbia, Okanagan Campus. Se presenta un método de muestreo no invasivo para recoger muestras de cabello de manera eficiente difícil pequeños mamíferos, como se muestra en la pika estadounidense. Demostrar la utilidad de este método de extracción de ADN de muestras de cabello y amplificar varios tipos de marcadores moleculares utilizados en los estudios de la ecología de la fauna silvestre y la conservación.

Video: A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression profiles of pyramidal neurons in layer III. It is well known that the cerebral cortex is an exceptionally heterogeneous structure comprising diverse regions, layers and cell types, each of which is characterized by distinct cellular and molecular compositions and therefore differential gene expression profiles. To circumvent the confounding effects of tissue heterogeneity, we used laser-capture microdissection (LCM) in order to isolate our specific cell-type i.e pyramidal neurons.
Approximately 500 pyramidal neurons stained with the Histogene staining solution were captured using the Arcturus XT LCM system. RNA was then isolated from captured cells and underwent two rounds of T7-based linear amplification using Arcturus/Molecular Devices kits. The Experion LabChip (Bio-Rad) gel and electropherogram indicated good quality a(m)RNA, with a transcript length extending past 600nt required for microarrays. The amount of mRNA obtained averaged 51&mu;g, with acceptable mean sample purity as indicated by the A260/280 ratio, of 2.5. Gene expression was profiled using the Human X3P GeneChip probe array from Affymetrix.

We describe a process using laser-capture microdissection to isolate and extract RNA from a homogeneous cell population, pyramidal neurons, in layer III of the superior temporal gyrus in postmortem human brains. We subsequently linearly amplify (T7-based) mRNA, and hybridize the sample to the Affymetrix human X3P microarray.

La obtención de ARN de alta calidad a partir de poblaciones de células individuales en tejido cerebral humano post-mortem

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression ...

Investigación

Biología

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the richness of biological information it provides. MR does not require any ionizing radiation and can noninvasively provide higher resolution and better signal-to-noise ratio in comparison to other tomographic or spectroscopic modalities. In this protocol, we will focus on small animal MR imaging and MR spectroscopy (MRI/MRS) to noninvasively acquire relaxation weighted 1H images of mouse and to obtain 31P spectra of mouse muscle. This work does not attempt to cover every aspect of small animal MRI/MRS but rather introduces basic procedures of mouse MRI/MRS experiments. The main goal of this work is to inform researchers of the basic procedures for in vivo MR experiments on small animals. The goal is to provide a better understanding of basic experimental procedures to allow researchers new to the MR field to better plan for non-MR components of their studies so that both MR and non-MR procedures are seamlessly integrated.

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

This work describes basic procedures of noninvasive small animal MRI and MRS in vivo.

No invasiva En Vivo Pequeños Animales MRI y MRS: Procedimientos básicos Experimental

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the ...

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested1. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue2. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.

Microarrays de ADN: Control de calidad de la muestra, la hibridación de la matriz y de escaneo

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, ...

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

La extracción de ADN de alto peso molecular a partir de tapetes microbianos

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

Generación de Plantilla ADN Immunoprecipitation cromatina alta calidad para la secuenciación de alto rendimiento (chip-ss)

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

Un manual de la pantalla pequeña molécula Acercarse De Alto Rendimiento Utilizando embriones de pez cebra

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and alopecia areata. Alopecia can also occur as a side effect of chemotherapy in cancer patients. In this study, our goal was to develop a consistent and reliable method to quantify hair loss in mice, which will allow investigators to accurately assess and compare new therapeutic approaches for these various forms of alopecia. The method utilizes a standard gel imager to obtain and process images of mice, measuring the light absorption, which occurs in rough proportion to the amount of black (or gray) hair on the mouse. Data that has been quantified in this fashion can then be analyzed using standard statistical techniques (i.e., ANOVA, T-test). This methodology was tested in mouse models of chemotherapy-induced alopecia, alopecia areata and alopecia from waxing. In this report, the detailed protocol is presented for performing these measurements, including validation data from C57BL/6 and C3H/HeJ strains of mice. This new technique offers a number of advantages, including relative simplicity of application, reliance on equipment which is readily available in most research laboratories, and applying an objective, quantitative assessment which is more robust than subjective evaluations. Improvements in quantification of hair growth in mice will improve study of alopecia models and facilitate evaluation of promising new therapies in preclinical studies.

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

Una nueva técnica de análisis cuantitativos de la pérdida del pelo en ratones Uso del análisis de escala de grises

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

A Web Tool for Generating High Quality Machine-readable Biological Pathways

PathWhiz is a web server built to facilitate the creation of colorful, interactive, visually pleasing pathway diagrams that are rich in biological information. The pathways generated by this online application are machine-readable and fully compatible with essentially all web-browsers and computer operating systems. It uses a specially developed, web-enabled pathway drawing interface that permits the selection and placement of different combinations of pre-drawn biological or biochemical entities to depict reactions, interactions, transport processes and binding events. This palette of entities consists of chemical compounds, proteins, nucleic acids, cellular membranes, subcellular structures, tissues, and organs. All of the visual elements in it can be interactively adjusted and customized. Furthermore, because this tool is a web server, all pathways and pathway elements are publicly accessible. This kind of pathway &#34;crowd sourcing&#34; means that PathWhiz already contains a large and rapidly growing collection of previously drawn pathways and pathway elements. Here we describe a protocol for the quick and easy creation of new pathways and the alteration of existing pathways. To further facilitate pathway editing and creation, the tool contains replication and propagation functions. The replication function allows existing pathways to be used as templates to create or edit new pathways. The propagation function allows one to take an existing pathway and automatically propagate it across different species. Pathways created with this tool can be &#34;re-styled&#34; into different formats (KEGG-like or text-book like), colored with different backgrounds, exported to BioPAX, SBGN-ML, SBML, or PWML data exchange formats, and downloaded as PNG or SVG images. The pathways can easily be incorporated into online databases, integrated into presentations, posters or publications, or used exclusively for online visualization and exploration. This protocol has been successfully applied to generate over 2,000 pathway diagrams, which are now found in many online databases including HMDB, DrugBank, SMPDB, and ECMDB.

PathWhiz is a comprehensive, online pathway drawing tool for generating biochemical and biological pathways. It uses publicly accessible databases and easily expandable palettes consisting of pre-drawn pathway components. This protocol describes how to easily build new pathways, replicate and edit existing pathways, and propagate previously drawn pathways to different organisms.

Una herramienta web para los procesos biológicos Generación de alta calidad legibles por máquina

PathWhiz is a web server built to facilitate the creation of colorful, interactive, visually pleasing pathway diagrams that are rich in biological ...

A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser capture microdissection (LCM) procedure. Since LCM studies work with low amounts of material, concerns about limited RNA yields are also important. Therefore, an LCM protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The effect of staining protocol, thickness of cryosections, microdissected tissue quantity, RNA extraction kit, and LCM system used on RNA yield and integrity obtained from microdissected bone cells was evaluated. Eight-&#181;m-thick frozen bone sections were made using an adhesive film and stained using a rapid protocol for a commercial LCM stain. The sample was sandwiched between a polyethylene terephthalate (PET) membrane and the adhesive film. An LCM system that uses gravity for sample collection and a column-based RNA extraction method were used to obtain high quality RNAs of sufficient yield. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study in situ gene expression in cells of any hard tissue in both physiological conditions and disease processes.

A laser capture microdissection (LCM) protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study gene expression in cells of any hard tissue.

Un protocolo de microdisección de captura láser que produce ARN de alta calidad a partir de huesos de ratón congelados

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Aislamiento de miocitos aurículas y ventriculares murinas de alta calidad para mediciones simultáneas de transitorios de Ca2+ y corriente de calcio tipo L

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...